Nucleic acid structure determination

Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes.

Biophysical methods

X-ray crystallography

X-ray crystallography is not common for nucleic acids alone, since neither DNA nor RNA readily form crystals. This is due to the greater degree of intrinsic disorder and dynamism in nucleic acid structures and the negatively charged (deoxy)ribose-phosphate backbones, which repel each other in close proximity. Therefore, crystallized nucleic acids tend to be complexed with a protein of interest to provide structural order and neutralize the negative charge.

Nuclear magnetic resonance spectroscopy (NMR)

Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. As of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy. Nucleic acid NMR uses similar techniques as protein NMR, but has several differences. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR, and because nucleic acid double helices are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are 1H or proton NMR, 13C NMR, 15N NMR, and 31P NMR. Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space. Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants, can be used to determine local structural features such as glycosidic bond angles, dihedral angles (using the Karplus equation), and sugar pucker conformations. For large-scale structure, these local parameters must be supplemented with other structural assumptions or models, because errors add up as the double helix is traversed, and unlike with proteins, the double helix does not have a compact interior and does not fold back upon itself. NMR is also useful for investigating nonstandard geometries such as bent helices, non-Watson–Crick basepairing, and coaxial stacking. It has been especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt complex conformations such as stem-loops and pseudoknots. NMR is also useful for probing the binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which resonances are shifted upon binding of the other molecule.

Cryogenic electron microscopy (cryo-EM) Cryogenic electron microscopy (cryo-EM) is a technique that uses an electron beam to image samples that have been cryogenically preserved in an aqueous solution. Liquid samples are pipetted on small metallic grids and plunged into a liquid ethane/propane solution which is kept extremely cold by a liquid nitrogen bath. Upon this freezing process, water molecules in the sample do not have enough time to form hexagonal lattices as found in ice, and therefore the sample is preserved in a glassy water-like state (also referred to as a vitrified ice), making these samples easier to image using the electron beam. An advantage of cryo-EM over x-ray crystallography is that the samples are preserved in their aqueous solution state and not perturbed by forming a crystal of the sample. One disadvantage, is that it is difficult to resolve nucleic acid or protein structures that are smaller than ~75 kilodaltons, partly due to the difficulty of having enough contrast to locate particles in this vitrified aqueous solution. Another disadvantage is that to attain atomic-level structure information about a sample requires taking many images (often referred to as electron micrographs) and averaging over those images in a process called single-particle reconstruction. This is a computationally intensive process. Cryo-EM is a newer, less perturbative version of transmission electron microscopy (TEM). It is less perturbative because the sample is not dried onto a surface, this drying process is often done in negative-stain TEM, and because Cryo-EM does not require contrast agent like heavy metal salts (e.g. uranyl acetate or phoshotungstic acid) which also may affect the structure of the biomolecule. Transmission electron microscopy, as a technique, utilizes the fact that samples interact with a beam of electrons and only parts of the sample that do not interact with the electron beam are allowed to 'transmit' onto the electron detection system. TEM, in general, has been a useful technique in determining nucleic acid structure since the 1960s. While double-stranded DNA (dsDNA) structure may not traditionally be considered structure, in the typical sense of alternating segments of single- and double-stranded regions, in reality, dsDNA is not simply a perfectly ordered double helix at every location of its length due to thermal fluctuations in the DNA and alternative structures that can form like g-quadruplexes. CryoEM of nucleic acid has been done on ribosomes, viral RNA, and single-stranded RNA structures within viruses. These studies have resolved structural features at different resolutions from the nucleobase level (2-3 angstroms) up to tertiary structure motifs (greater than a nanometer).

Chemical probing

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